The present invention relates to a nucleotide sequence, called xe2x80x9cpolyribozymexe2x80x9d, capable of conferring on plants resistance to viruses, as well as a process for making the plants resistant. The invention also relates to the plants expressing the polyribozyme.
Several approaches have been developed to confer on cultivated plants resistance to viruses by integrating into the genome of the plants viral nucleic acid sequences: the gene for the capsid protein, the genes for non-structural proteins, anti-sense viral RNA sequences and RNAs of satellite viruses (see, for example, Cuozzo et al., 1988, Bio/Technology 6, 549-557; Rezaian et al., 1988, Plant. Mol. Biol., 11, 463-471; Harrison et al., 1987, Nature 328, 797-802).
These publications report the production of partial resistances or tolerances. Nonetheless, in most cases there are delayed symptoms or attenuated symptoms but not complete resistance.
Furthermore, some of these procedures, for example those employing the RNAs of satellite viruses, can give rise to new problems. For example, a satellite virus which reduces symptoms in one species may become lethal for another species. Moreover, mutations in the nucleotide sequences of the satellite virus introduced into the plants may increase the severity of the infection instead of diminishing it.
Similarly, the use of the capsid protein to confer resistance has disadvantages. For example, the capsid protein of a particular strain of the virus does not necessarily protect the plant against an infection by another strain of the virus. It is difficult to use the degree of homology of the amino acid sequence of the capsid protein between different viruses or between different strains to predict the degree of tolerance allowed by the expression of the protein. Furthermore, the expression of capsid proteins to protect against viral infection presents the risk of inducing heteroencapsidation between the capsid protein expressed in the plant and other viruses infecting the transgenic plant. Although it has never been demonstrated for transgenic plants, this heteroencapsidation has already been observed between two strains of BYDV and between ZYMV and PRSV.
The use of ribozymes has also been considered for conferring on plants resistance to viruses. Ribozymes are RNA molecules which act as enzymes by specifically catalysing the cleavage of the target RNA. The first experiments with ribozymes in plant cells were described in the patent application EP-A-321021. Since then, several authors have tried to optimise the structure of the ribozyme and the operating conditions in order to obtain efficient cleavage of the viral RNA.
For example, Lamb and Hay (J. Gen. Virol., 1990, 71:2257-2264) have demonstrated the in vitro cleavage by mono-ribozymes of the RNA of the Potato Leaf Roll virus (PLRV) in regions coding for the RNA polymerase and the capsid protein. However, the in vitro cleavage reaction only occurs at 40xc2x0 C.; it has not been possible to observe any reaction at all at 0xc2x0 C. Plants are usually cultivated between 10 and 30xc2x0 C., depending on the species. Thus, for in vivo use, Lamb and Hay suggest that the length of the complementary arms be increased. But, if the arms are too long, the formation of a stable duplex between the target RNA and the ribozyme can be caused, preventing the dissociation of the ribozyme and making it incapable of catalysing another cleavage reaction. Furthermore, depending on the length and sequence of the complementary arms, the ribozyme itself may form secondary structures which diminish its cleavage activity.
Edington and Nelson (xe2x80x9cGene Regulation: Biology of Antisense RNA and DNA: Ed. ERICKSON and IZANT, Raven Press Ltd, New-York, 1992) have described the in vitro and in vivo use of mono- ribozymes to inactivate the polymerase gene of the Tobacco Mosaic virus (TMV). They observed that the ribozymes exhibited a very different behaviour depending on whether they were used in vitro or in vivo. The activity of a ribozyme in vitro can not thus be used to predict the activity of the same ribozyme in vivo. For example, in vitro cleavage appear to be of low efficiency and requires a ribozyme concentration 20 times higher than the concentration of the TMV genomic RNA. On the other hand, in an in vivo experiment using tobacco protoplasts infected by TMV, the ribozyme suppresses 90% of the multiplication of the viral RNA. It is interesting to note that the anti-sense RNA used as control only inhibits 20% of the viral multiplication. These workers also refer to the studies of Gerlach et al. who made use of a polyribozyme targeted against the gene for the polymerase of TMV. This polyribozyme did not function in vitro owing to the length of the duplex formed between the ribozyme and the target RNA. On the other hand, in vivo, this polyribozyme cleaved the substrate. Transgenic tobacco plants expressing either the monoribozyme or the polyribozyme have shown a delay of symptoms after infection by the TMV. Complete resistance, i.e. the definitive absence of symptoms, is not described. The authors conclude that the parameters such as the optimal length of the complementary arms, the choice of the target sequence and the choice of the promoter, enabling possible problems of xe2x80x9ccompartment-alisationxe2x80x9d of the ribozymes to be overcome, must be determined by experiment.
EP-A-0421376 describes ribozymes directed against a non-coding RNA sequence of CMV. WO-A-9213090 describes the inactivation of the RNA of the capsid protein of the CMV by the introduction of a heterologous sequence within the sequence using a monoribozyme of the xe2x80x9cGroup I intronxe2x80x9d type. None of these documents describes the production of complete resistance to the CMV.
The technical problem which the present invention proposes to resolve is to provide a reliable agent, devoid of disadvantages, for conferring on plants resistance to viruses.
The present inventors have resolved this problem by the conception and use of a polyribozyme directed against the capsid protein of a virus. This polyribozyme is capable of inactivating the gene coding for this protein, and of thus conferring complete resistance to viruses.
The efficiency of the polyribozyme of the invention is surprising in the light of the mediocre results obtained in the prior art with the anti-sense sequences of the gene for the capsid protein, since each of these procedures involves an inactivation of the corresponding RNA. In addition, several authors had advised against the use of trans acting polyribozymes because the ribozymes are unable to function independently of each other and because catalytic regions having identical sequences sometimes have a tendency to hybridize to each other, which leads to inactive structures (see, for example, Taira, HFSP. Workshop xe2x80x9cRNA- Editingxe2x80x94Plant Mitochondriaxe2x80x9d, Abstract Book, Berlin, Sep. 15-20, 1992). The results obtained according to the invention are unexpected in view of the target selected, on the one hand, i.e. the capsid protein and, on the other hand, the method used to inactivate the target, i.e. a polyribozyme.
In addition to the efficiency of the inactivation, the polyribozymes of the invention possess a number of advantages in comparison with known procedures:
The ribozymes function as enzymes, catalysing the cleavage of several viral RNAs specifically without modification of structure. This enzymatic cleavage leads to the destruction of all of the viral RNAs whereas the expression of the capsid protein which inhibits viral infection functions as an inhibitor of viral multiplication.
The ribozymes are non-coding RNA molecules which can not induce heteroencapsidations or generate new viral strains.
Whereas the specificity of the tolerance induced by the capsid protein is difficult to predict, ribozymes can be constructed in order to cleave specifically one or more viral strains, or several related viruses if the complementary arms correspond to the regions of homology conserved between the different strains or between the different related viruses.
In order to have a complete understanding of the invention, it will be useful to specify certain facts concerning ribozymes in general. A ribozyme is an RNA molecule which, by virtue of its sequence and secondary structure, possesses an endoribonuclease activity which enables it, when it hybridizes with a second molecule of complementary RNA, to cleave this second RNA. This latter is hence a xe2x80x9csubstratexe2x80x9d for the ribozyme.
The ribozyme has two essential parts:
(i) a sequence, which will be called xe2x80x9ccomplementary sequencexe2x80x9d in what follows, and which is selected so that, it is complementary to the substrate which it is desired to cleave, this enabling the two molecules to hybridize;
(ii) and a catalytic region which has a conserved sequence irrespective of the substrate selected and which does not take part in the hybridization with the substrate on account of its secondary structure which is in the form of a xe2x80x9cloopxe2x80x9d.
Usually, the catalytic region is located within the complementary sequence, one part of the complementary sequence thus being situated at the 5xe2x80x2 of the catalytic region and the other part at the 3xe2x80x2. These fragments of the complementary sequence on each side of the catalytic region are often called xe2x80x9chybridizing armsxe2x80x9d.
The object of the present invention is a polyribozyme. More particularly, it is a nucleic acid sequence, called xe2x80x9cpolyribozymexe2x80x9d, which has an endoribonuclease activity and is capable of inactivating the gene for the capsid protein of a virus, characterized in that it comprises:
i) a sequence complementary to at least a part of the gene or its transcript or its replication intermediates and, included at distinct sites in this complementary sequence:
ii) a plurality of ribozyme catalytic regions;
iii) and, optionally, one or more sequences not complementary to the transcript of the said gene, the said non-complementary sequence(s) being inserted between 2 consecutive bases of the complementary sequence.
The term xe2x80x9cpolyribozymexe2x80x9d in the context of the present invention means an RNA molecule constituted by a head-to-tail series of ribozymes, the ribozyme thus being the unit motif of the polyribozyme. In other words, it is a series of catalytic regions connected together by hybridizing arms, the total length of these arms constituting the complementary sequence. The polyribozyme normally acts as a xe2x80x9cuni-moleculexe2x80x9d against a single transcript, i.e. the cleavage sites of each of the catalytic regions are located on the same transcript: the capsid protein. The polyribozyme of the invention may also comprise, in addition to the 2 essential parts [(i), complementary sequence] and [(ii), catalytic regions] described above, one or more sequences (iii) non-complementary to the substrate. The nature and function of these non-complementary sequences will be described in detail hereafter.
Of the 2 essential parts of the polyribozyme, the complementary sequence is that which determines the substrate. In the case of the present invention, it is a sequence complementary to the gene for the capsid protein of a virus, or to a fragment of this gene. When the virus is an RNA (+) virus, the genes of which serve directly as mRNA, the complementary sequence is really complementary to the gene. In other cases, it is complementary to the transcript of the gene. It may also be complementary to a replication intermediate.
The complementary sequence may hybridize with the entire length of the capsid gene. In this case, the total length of the complementary sequence varies as a function of the length of the capsid gene in question. On the other hand, the complementary sequence may hybridize with only a fragment of the gene. The fragment in question must be long enough to allow the inclusion of at least two catalytic regions in the corresponding sequence of the polyribozyme. In general, the length of the complementary sequence, not counting the catalytic regions (i.e. the sum of the hybridizing arms), may vary from about 40 to 2000 bases. A length of 400 to 1000 is preferred, very many viruses having a gene for the capsid protein of about 1000 bases (for example, CMV, PLRV).
The term xe2x80x9ccomplementaryxe2x80x9d in the context of the invention means a sufficiently high degree of complementarity to allow stable hybridization between the polyribozyme and this substrate, and the efficient cleavage of the substrate. When the polyribozyme does not contain a sequence of type (iii), i.e. xe2x80x9cnon-complementaryxe2x80x9d, the degree of complementarity is usually 100%. The presence of a certain number of mismatches in the sequence, for example up to 10%, may be tolerated provided that that does not prevent the hybridization and cleavage of the substrate.
The (ii) part of the polyribozyme, i.e. the catalytic region, is derived from any type of suitable ribozyme, for example xe2x80x9chammer headxe2x80x9d, xe2x80x9chairpinxe2x80x9d or xe2x80x9cgroup I intronxe2x80x9d. One and the same polyribozyme may contain catalytic regions derived from different types of ribozymes, for example, xe2x80x9chammer headxe2x80x9d and hairpinxe2x80x9d. Catalytic regions are preferably derived from ribozymes of the xe2x80x9chammer headxe2x80x9d type, the consensus structure of which is illustrated in the FIGS. 1A, B, C and D. These ribozymes are described in detail in the patent application EP-A-321021 and WO-A-9119789.
Although the catalytic regions illustrated in FIG. 1 have a conserved structure and sequence, it has been observed that some nucleotides may be deleted, inserted, substituted or modified without prejudice to the activity of the ribozyme. The invention comprises the use of these modified catalytic regions in the polyribozyme provided that their catalytic activity is conserved. This activity can be verified by using the tests described below.
For example, one or more nucleotides of the catalytic region II illustrated in FIG. 1A may be replaced by nucleotides containing bases such as adenine, guanine, cytosine, methylcytosine, uracil, thymine, xanthine, hypoxanthine, inosine or other methylated bases. The xe2x80x9cconservedxe2x80x9d bases C-G which together form the first base pair of the catalytic loop, can be replaced by U-A (Koizumi et al., FEBS Letts. 228, 2, 228-230, 1988).
The nucleotides of the catalytic region illustrated in FIG. 1 can also be modified chemically. The nucleotides are composed of a base, a sugar and a monophosphate group. Each of these groups can thus be modified. Such modifications are described in xe2x80x9cPrinciples of Nucleic Acid Structurexe2x80x9d (Ed. Wolfram Sanger, Springer Verlag, N.Y., 1984). For example, the bases may bear substituents such as halogeno, hydroxy, amino, alkyl, azido, nitro, phenyl groups, etc. The sugar moiety of the nucleotide may also be subjected to modifications such as the replacement of the secondary hydroxyl groups by halogeno, amino or azido groups or even to 2xe2x80x2 methylation.
The phosphate group of the nucleotides may be modified by the replacement of an oxygen by N, S or C, giving rise to a phosphoramidate, phosphorothioate and phosphonate, respectively. These latter may exhibit useful pharmacokinetic properties.
The bases and/or the nucleotides of the catalytic region may also bear substituents such as amino acids, for example, tyrosine or histidine.
It has also been observed that additional nucleotides may be inserted at certain sites of the catalytic region without prejudice to the activity of the ribozyme. For example, an additional base selected from among A, G, C or U may be inserted after A1 in FIG. 1A or 1B.
According to a variant of the invention, the ribozyme may comprise as catalytic region one or more structures such as those illustrated in FIG. 1D. This structure, called xe2x80x9cminizymexe2x80x9d, is described in the international patent application WO-A-9119789. It represents a catalytic region of the xe2x80x9chammerheadxe2x80x9d type, the xe2x80x9cloopxe2x80x9d of which has been replaced by a xe2x80x9cPxe2x80x9d group. P may be a covalent link between G and 1A, one or more nucleotides (RNA or DNA, or a mixture, or even derivatives described above) or any atom or group of atoms other than a nucleotide which does not affect the catalytic activity. When P represents a plurality of nucleotides, it may contain internal base pairings. The sequence and the number of nucleotides constituting the group xe2x80x9cPxe2x80x9d is not critical and may vary from 1 to 20 nucleotides for example, and preferably from 1 to 6. It is preferable to select a sequence lacking internal base pairings of the Watson-Crick type.
The catalytic activity of the polyribozymes of the invention may be verified in vitro by placing the polyribozyme, or a sequence which after transcription will give rise to the polyribozyme, in contact with the substrate, followed by demonstration of the cleavage. The experimental conditions for the in vitro cleavage reaction are the following: a temperature comprised between 4 and 60xc2x0 C., and preferably between 20 and 55xc2x0 C., a pH comprised between about 7.0 and 9.0, in the presence of divalent metals, such as Mg2+, at a concentration of 1 to 100 mM (preferably 1 to 20 mM). The polyribozyme is usually present in an equimolar ratio with the substrate, or in excess. The in vitro cleavage reactions are advantageously carried out according to the procedure described by Lamb and Hay (J. Gen. Virol., 1990, 71, 2257-2264). This article also describes suitable conditions for in vitro transcription for the production of ribozymes from oligodeoxyribonucleotides inserted into plasmids.
The in vivo cleavage conditions are those existing naturally in the cell.
The xe2x80x9chammerheadxe2x80x9d ribozymes cleave the substrate immediately downstream from a xe2x80x9ctargetxe2x80x9d site XXX, preferably XUX, in which X represents one of the 4 bases A, C, G, U and U represents uracil. One particularly preferred target sequence is XUY in which Y represents A, C or U and Xis often G, for example, GUC. Other target sites are possible, but less efficient, for example CAC, UAC and AAC. In the case of the ribozymes of the xe2x80x9chairpinxe2x80x9d type, a preferred target sequence is AGUC.
These target sequences are important in the construction and functioning of the polyribozymes, not only because they indicate the positions of cleavage of the substrate but also because they define the position at which the catalytic region must be inserted in the complementary sequence. In fact, each catalytic region of the polyribozyme must be situated at a site in the complementary sequence which corresponds to a XUX site of the transcript. For example, if one XUX site is situated at position 108 of the gene for the capsid protein and another at position 205, a catalytic region is inserted at the corresponding position at 108 in the complementary sequence and another at 205.
The motif XUX is a motif which occurs very frequently in the RNA sequences. For example, on average there is a GUC motif every 64 bases in a sequence having a random and equal distribution of bases. This signifies that the substrate usually contains a plurality of XUX cleavage sites. As indicated above, the catalytic regions of the polyribozymes are situated at positions of the complementary sequence which correspond to the XUX sites. However, it is not necessary to include a catalytic region for each XUX target sequence of the substrate in order to obtain an efficient cleavage according to the invention. According to the invention, an efficient cleavage is obtained when the polyribozyme contains at least 2 catalytic regions. The total number of catalytic regions included in the complementary sequence is equal to or smaller than the total number of XUX sites present in the gene. The polyribozyme of the invention may thus contain a very variable number of catalytic regions. For example, in the case of CMV, the polyribozyme may contain from about 2 to about 11 or 12 catalytic regions, when the target sequence is GUC. In the case in which it is decided to include a smaller number of catalytic regions in the complementary sequence than the number of XUX sites in the substrate, the choice of the sites selected may be made by respecting the following criteria:
a) the distance between the 2 XUX sites targeted and, consequently, between 2 catalytic regions in the polyribozyme must be long enough to enable the hybridizing arms of the polyribozyme situated between the corresponding catalytic regions to hybridize with the substrate in a stable manner and to prevent the catalytic regions hybridizing with themselves. A distance of at least 8 bases, and preferably at least 14 bases, for example about 20 bases is particularly advantageous. Of course, this criterion must only be taken into consideration when the substrate contains XUX sites very close together. Otherwise, if the XUX sites of the substrate are separated from each other by more than 8 to 20 bases, this selection criterion is not important.
b) the XUX sites targeted are preferably situated in a part of the gene for the capsid protein which does not have significant secondary structure. This facilitates the access of the polyribozyme to the substrate and increases its efficacy.
c) the XUX sites targeted may form part of the regions of homology conserved between different strains of one and the same virus, or between different related viruses. The polyribozymes constructed by respecting this criterion may cleave specifically several viral strains or several related viruses. For example, the central region of the gene for the capsid protein of the PLRV is highly conserved compared with sequences of the capsid proteins of the related viruses BWYV and BYDV. The XUX sites, and particularly GUX within this central region, thus constitute preferred sites for a polyribozyme according to this variant of the invention.
Also, by way of example, the 5xe2x80x2 end (over a length of about 100 bases) of the sequence of the capsid protein of the CMV is highly conserved between the strains I17F, FNY, M, I, O, Y, D and C. At position 84 within this conserved sequence there is a conserved GUC site in all of these strains.
According to this variant of the invention, a polyribozyme capable of inactivating several strains of the CMV comprises among its catalytic regions one catalytic region which is situated at the site of the complementary sequence corresponding to the position 84. (see examples hereafter).
d) another selection criterion of the XUX sites targeted is the absence of homology with endogenous genes of the plant to be transformed. In fact, although they are rare, some viruses possess sequences which find a homology in the genome of plants. It is thus important to avoid XUX sites situated within such a sequence.
According to a particularly preferred embodiment of the invention, the polyribozyme may comprise, in addition to the 2 essential parts (i) and (ii) described above, a 3rd constituent (iii) which is one or more sequences non-complementary to the gene for the capsid protein of the virus. Like the catalytic regions, these non-complementary sequences are inserted at distinct sites of the complementary sequence, the complementarity being interrupted by the insertion. Surprisingly, it was observed by the inventors that the presence of such non-complementary sequences within the hybridizing arms of the polyribozyme does not prevent the hybridization of the polyribozyme with the substrate, and in some cases can even improve the efficiency of the cleavage reaction.
These non-complementary sequences are inserted between 2 consecutive bases of the complementary sequence, the non-complementary sequence thus forming a colinear insertion with the complementary sequence. In this case, the polyribozyme has the structure:
((hybridizing arm-catalytic region-hybridizing arm)xe2x88x92(non-complementary sequence)n)p
in which n=0 or 1, and p greater than 1.
As an example of this embodiment of the invention, mention may be made of a polyribozyme composed of a sequence of ribozymes, the hybridizing arms of which are complementary to distinct fragments, consecutive and adjoining, of the substrate and which are connected together by non-complementary sequences. In other words, the aggregate of the hybridizing arms in such a structure reconstitute the sequence complementary to the gene for the capsid protein.
The presence of non-complementary sequences in the polyribozyme signifies that the distance between two catalytic regions of the polyribozyme is greater than the distance between two corresponding GUC sites in the substrate. According to this variant of the invention, the length of the hybridizing arms located on each side of a catalytic region must be at least 4 bases, and preferably at least 8 bases on each side and may be as many as 800 to 1000 bases.
The nature of the non-complementary sequence(s) may be very variable depending on its (their) function. There may be sequences which have a xe2x80x9cpaddingxe2x80x9d function, i.e. which serve to increase the distance between two catalytic regions of the polyribozyme, when the corresponding two XUX sites of the substrate are relatively close to each other. In this manner, the formation of inactive duplexes between two neighbouring catalytic regions can be avoided. It is also possible to use as non-complementary sequences, sequences which have a defined secondary structure, which may have the effect of preventing a polyribozyme of considerable length, for example one with more than 800 bases, from refolding on itself in an inactive secondary structure. As an example of this type of structure, mention may be made of a ribozyme rendered inactive by the deletion of one or more essential bases. This mode of embodiment of the invention is exemplified by the polyribozyme 136 described in the examples below.
The non-complementary sequence of the polyribozyme may also have a precise function, for example, it may be constituted by a coding sequence which can be used to select transformants or also a sequence containing a ribozyme which acts on a substrate other than the capsid protein or which is cis acting on a part of the polyribozyme. Generally speaking, the non-complementary sequence does not code for a protein. It may also contain multisites for cloning. The non-complementary sequence usually has a length comprised between 2 and 500 bases, for example 20 to 100 bases. When there is a plurality of complementary sequences, they may together constitute up to about 90% of the length of the polyribozyme, for example 50%.
The polyribozyme of the invention is usually constituted of RNA. Nonetheless,it is possible to replace some parts of the polyribozyme by DNA, for example the hybridizing arms or parts of these arms, or also a part of the catalytic region, in particular the xe2x80x9cloopxe2x80x9d, provided that the catalytic activity is maintained (see, for example, the substitution of the RNA by DNA described in the international patent application WO-9119789).
The polyribozyme of the invention can be constructed to inactivate any viral capsid protein. The capsid protein is the protein sub-unit, coded by the viral genome, which makes up the polymeric capsid. The capsid is composed of a succession of these identical protein sub-units which line-up along the nucleic acid. The spatial arrangement of the capsid sub-units gives rise to either a helicoidal or an icosahedric structure, according to the virus. The invention concerns polyribozymes directed to the capsid proteins of viruses having either helicoidal particles, or icosahedric particles, as well as those having an envelope. The envelope is a lipoprotein membrane surrounding the nucleocapsid.
As an example of a suitable virus, mention may be made of a virus selected from the following groups: the caulimoviruses, for example the Cauliflower Mosaic Virus (CaMV); the Geminiviruses, for example the Maize Streak Virus (MSV); the Reoviridae, for example the Wound Tumor Virus (WTV); the Rhabdoviridae, for example the Potato Yellow Dwarf Virus (PYDV), the Tomato Spotted Wilt Virus (TSWV); the Tobamoviruses, for example the Tobacco Mosaic Virus (TMV); the Potexviruses, for example the Potato Virus X (PVX); the Potyviruses, for example the Potato Virus Y (PVY); the Carlaviruses, for example the Carnation Latent Virus (CLV); the Closteroviruses, for example the Beet Yellow Virus (BYV); the Tobraviruses, for example the Tobacco Rattle Virus (TRV); the Hordei-viruses, for example the Barley Stripe Mosaic Virus; the Tymoviruses, for example the Turnip Yellow Mosaic virus (TYMV); the Luteoviruses, for example the Barley Yellow Dwarf Virus (BYDV) or the Potato Leaf Roll Virus (PLRV); the Tombusviruses, for example the Tomato Bushy Stunt Virus (TBSV); the Sobemoviruses, for example the Southern Bean Mosaic Virus (SBMV); the Tobacco Necrosis virus (TNV); the Nepoviruses, for example the Tobacco Ring Spot Virus (TRSV); the Comoviruses, for example the Cow Pea Mosaic Virus (CPMV); the Pea Enation Mosaic Virus (PEMV); the Cucumoviruses, for example the Cucumber Mosaic Virus (CMV); the Bromoviruses, for example the Brome Mosaic Virus (BMV); the Ilarviruses, for example the Tobacco Streak Virus (TSV). the sequences of these proteins are known (see for example the numerous literature references cited in the monograph: xe2x80x9cElxc3xa9ments de Virologie Vxc3xa9gxc3xa9talexe2x80x9d, Pierre Cornuet, I. N. R. A. Paris, 1987, ISBN: 2-85340-808-6).
According to a particularly preferred variant, the capsid protein is that of the Cucumber Mosaic Virus (CMV). The Cucumber Mosaic Virus is a virus belonging to the group of the Cucumoviruses which are of great agronomic importance since more than 750 species of plants may be infected by the CMV. The CMV is a multi-component virus composed of icosahedral particles containing three genomic RNAs (RNAs 1 to 3) and a subgenomic RNA (RNA 4). The RNA 3 contains a copy of the gene for the capsid protein; however, the subgenomic RNA4, which is derived from RNA 3, serves as matrix for the synthesis of the capsid protein. The different strains of CMV are divided into two groups:
the sub-group I which comprises the strains C, D, FNY, Y, I17F and Chi;
the sub-group II to which the strains Q and WL belong.
The comparison of the amino acid sequences of the capsid proteins of the CMV strains belonging to the same sub-group shows a homology of 95%. The sequence homologies between the sub-groups I and II are lower, of the order of 80%.
The polyribozymes of the invention directed against the capsid protein of the CMV (strain I17F) have been found to be extremely efficient in inactivating the different strains of the CMV, and have resulted in complete resistance of the transformed plants.
In addition to the polyribozymes, the invention also relates to a process for making a plant resistant to a virus, characterized by the introduction into the plant of a polyribozyme or a sequence coding for a polyribozyme such as described above.
Usually, the introduction of the polyribozyme into the plant is performed by genetic transformation, a DNA sequence coding for the polyribozyme thus being integrated stably into the genome of the plant.
All of the known means for introducing foreign DNA into plants may be used, for example Agrobacterium, electroporation, protoplast fusion, bombardment with a particle gun, or penetration of DNA into cells such as pollen, the microspore, the seed and the immature embryo, viral vectors such as the Geminiviruses or the satelite viruses. Agrobacterium tumefaciens and rhizogenes constitute the preferred means. In this case, the sequence coding for the polyribozyme is introduced into a suitable vector together with all of the regulatory sequences necessary such as promoters, terminators, etc. . . . as well as any sequence necessary for selecting the transformants.
The invention also relates to the transgenic plants obtained by the process. More particularly, it relates to transgenic plants resistant to a virus, characterized in that they contain in their genome a sequence which, after transcription, gives rise to a polyribozyme according to the invention .
In the context of the invention, the term xe2x80x9ccomplete resistancexe2x80x9d signifies a complete absence of symptoms; xe2x80x9ctolerancexe2x80x9d signifies that the plant is infected, i.e. it shows symptoms, but subsequently recovers. The term xe2x80x9csensitivexe2x80x9d signifies that the plant exhibits symptoms and replicates the virus. The expression xe2x80x9cresistant typexe2x80x9d refers to the sum of the completely resistant plants and the tolerant plants.
The xe2x80x9cresistantxe2x80x9d nature of the transgenic plants of the invention can be tested in the following manner: a self-fertilization, or a cross with a non-transformed genotype, is carried out on a primary descendant to obtain T1. Subsequently, T1 plants is inoculated with the virus in question. According to the invention, after self-fertilization 75% of the T1 are completely resistant. In the case of a cross with a non-transformed genotype, 50% of the plants are completely resistant (these figures are obtained, according to the invention, by testing a whole population of plants which had been subjected to a transformation and regeneration procedure. It is to be noted that only 75% of these plants are transformed).
The transformed nature of a plant can be verified by performing a xe2x80x9cSouthern blotxe2x80x9d analysis and the expression of the sequence introduced by the transformation is verified by carrying out a xe2x80x9cNorthern blotxe2x80x9d analysis. These analyses are described in the examples which follow.
The methods of transformation and regeneration of plants known in the prior art lend themselves perfectly to the production of transgenic plants protected by the polyribozyme of the invention. As an example, mention may be made of the method of transformation and regeneration of the melon described in the patent application No. EP-A-0412912.
The transgenic plants resistant to the CMV are particularly preferred, for example the melon, the cucumber, the courgette, the tomato, the pepper, the bean.